Today, an analysis of the nucleotide sequence of a plant is rapid due to advancing sequencing methods and cost reduction. Among Cucurbitaceae plants, recently, full-length sequences of cucumber (2009), melon (2012), and watermelon (2013) were disclosed, but, in construction of a transformant, there is a difficulty in conducting a study on gene function through reverse or forward genetics due to a low success rate and inadequate development of a tool for a gene function analysis. To solve such a problem, viral vectors that can be studied are being developed. While viral vectors capable of being applied to other crops such as tomatoes, peppers, beans, etc. have been developed over time, the only choice for Cucurbitaceae plants is Apple latent stunt virus, which was developed in Japan in 2009. A vector constructed to express a heterologous protein in plants based on Tobamovirus, which infects cucurbits, is the Cucumber green mottle mosaic virus (CGMMV), which had an expression result without a prediction of a subgenomic RNA promoter (SGP) with a small-sized epitope attached behind the coat protein. Therefore, to achieve gene silencing and stable heterologous protein expression, an attempt was made to construct a vector by isolating and identifying CFMMV using Cucurbitaceae plants as main hosts. CFMMV is a member of the genus Tobamovirus, for which crops such as Cucurbitaceae plants including cucumbers, pumpkins, melons, oriental melons, watermelons, gourds, and tobacco plants are hosts.
The inventors submitted the full-length sequence of CFMMV that had been previously isolated and identified to the NCBI Genbank (Accession no. JN226146) and also received a patent for a recombinant clone of a CFMMV-derived attenuative virus (10-1342084). The present invention is for expressing the whole or a part of an insert in a host plant by inserting an innate gene of the host plant through artificial modification of the nucleotide sequence of CFMMV or inserting a gene of a foreign organism.
Tobamovirus is characterized by having a single genome, whose subgroups include Solanaceae-, Cruciferae- and Cucurbitaceae-infectious Tobamoviruses, and recently, Malvaceare-infectious and Cucurbitaceae-infectious Tobamoviruses have been reported. Development and research on viral vectors using tobacco mosaic virus (TMV), which is a representative Tobamovirus, and tomato mosaic virus (ToMV), tobacco mottle green mosaic virus (TMGMV) and sunn-hemp mosaic virus (SHMV) have been successfully performed in tobacco and tomato.
In the development of viral vectors, it is important to select an insertion position of a multiple cloning site (MCS), and, following the position selection, subgenomic promoter (SGP) mapping should be preferentially performed.
The inventors intend to provide a host plant-infectious viral vector that can be used in studying a gene function through gene silencing in Cucurbitaceae plants, which is difficult, and can also in vivo express a heterologous protein in plants.